Pediatric Pulmonology
Val66Met Polymorphism in the BDNF Gene in Children with Bronchial Asthma Milos Jesenak, MD, PhD,1 Eva Babusikova, MSc, PhD,2 Andrea Evinova, MSc, PhD,2 Peter Banovcin, MD, PhD,1 and Dusan Dobrota, MD, PhD2 Summary. Objectives: Bronchial asthma is a chronic respiratory disease characterized by airway inflammation. There is increasing evidence that neurotrophins play an important role in the development and maintenance of neurogenic airway inflammation in chronic allergic diseases. Working Hypothesis: Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and has several important functions in the airways. There are only a few reports on the association between genetic variations in the BDNF gene and various allergic diseases, and the results are generally conflicting. Therefore, we aimed to study the functional polymorphism Val66Met (also called rs6265 or G196A) in the BDNF gene in a group of asthmatic children and healthy controls. Study Design, Patient-Selection, and Methodology: We studied 248 asthmatic patients (aged 12.28 0.24 years) and 249 healthy children (aged 13.14 0.48 years). Analysis of the Val66Met polymorphism of the BDNF gene was performed by polymerase chain reaction (PCR) and PCR products were digested by PmlI. Results: The prevalence of the Val66Met polymorphisms (Val/Val, Val/Met, and Met/Met) was 61.7%, 33.5%, and 4.8% in asthmatics, respectively, and 47.0%, 51.8%, and 1.2% in healthy subjects, respectively. We observed a significant association of the Met/Met variant genotype with asthmatics (OR ¼ 4.17, 95% CI ¼ 1.16–14.96, P ¼ 0.018). The Val/Met genotype was protective against bronchial asthma (OR ¼ 0.69, 95% CI ¼ 0.48–0.99, P ¼ 0.045), especially in girls (OR ¼ 0.34, 95% CI ¼ 0.20–0.59, P ¼ 0.001). Conclusion: Specific BDNF gene polymorphism may contribute to bronchial asthma susceptibility. Our study suggested the positive association between selected functional BDNF ß 2014 Wiley Periodicals, Inc. polymorphism (rs6265) and asthma in children. Pediatr Pulmonol. Key words: brain-derived neurotrophic factor; bronchial asthma; children; neurogenic inflammation; neurotrophins; gene polymorphism. Funding source: Ministry of Education, Science, Research and Sport of the Slovak Republic VEGA; Number: 1/0071/11.
INTRODUCTION
Bronchial asthma is the most frequent chronic respiratory disease in children and is clinically characterized by airway inflammation, airway hyperresponsiveness, and reversible airflow limitations. However, the links between airway inflammation and changes in pulmonary functions are not completely understood. There is increasing evidence that one of the most important pathogenetic aspects responsible for the induction, maintenance, and progress of the airway changes in asthmatic patients is neurogenic inflammation.1 While there is convincing evidence of neurogenic inflammation in animal models of asthma,2 the evidence in humans is less clear and different studies brought conflicting and inconsistence results.3 Research was focused on the concentration of different neurotrophins and their function in the airways, but more recently, the association studies between different polymorphisms in the neurotrophins’ genes were also conducted. Neurotrophins play a critical role in the pathogenesis of allergic inflammation in asthma.4,5 ß 2014 Wiley Periodicals, Inc.
There is increasing evidence of the various members of the neurotrophin family participating in the development and
1 Department of Paediatrics, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia. 2 Department of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia.
Conflict of interest: None.
Correspondence to: Assoc. Prof. Eva Babusikova, MSc, PhD, Department of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Mala Hora 4, 036 01 Martin, Slovakia. E-mail: [email protected] Received 6 May 2013; Accepted 21 April 2014. DOI 10.1002/ppul.23065 Published online in Wiley Online Library (wileyonlinelibrary.com).
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maintenance of airway inflammation. There are several studies confirming the involvement of nerve growth factor in bronchial asthma,6–8 but the studies with brain-derived neurotrophic factor (BDNF) showed conflicting results regarding both the association between BDNF concentration or selected polymorphisms in asthmatic patients. In an animal model of allergic asthma, the up-regulation of BDNF in bronchoalveolar lavage fluid (BALF) and airway infiltrates after allergen challenge in sensitized mouse was detected.9 Up-regulation of neurotrophins including BDNF has also been demonstrated in asthmatics following segmental allergen provocation.10 Most of the vagal neurons innervating the lung are BDNF-dependent.11 Therefore, the local production of BDNF in allergic inflammation may be involved in neuronal changes occurring in bronchial asthma. Several authors have suggested that BDNF may serve as a mediator, linking airway inflammation with the neuronal changes observed in bronchial asthma. BDNF is a key mediator of neuronal plasticity, which significantly contributes to airway obstruction and hyperresponsiveness. Inhibition of endogenous BDNF reduces enhanced airway tone and neuronal hyperreactivity in allergen-challenged animals, while the administration of recombinant BDNF induces these asthmatic changes in healthy animals.9 Another possible mechanism of BDNF action in asthma is its stimulatory effect on the production and release of different tachykinins (e.g., substance P and neurokinin A), which are other very important mediators of asthma.12,13 BDNF enhances the proliferation of airway smooth muscles, which can contribute to the remodeling of the airways in chronic respiratory diseases.14 The expression of BDNF was shown to be induced also by cigarette smoke followed by the changes in the signaling in airway smooth muscle, which then contribute to cigarette smoke-induced airway hyperresponsiveness.15 Moreover, BDNF is known to elicit neuropeptide synthesis in neurons and support the survival, differentiation and function of a broad spectrum of neurons of the central and peripheral nervous system. At the clinical level, subjects with moderate to severe asthma have higher BDNF levels than mild asthmatics and controls16; therefore, BDNF could serve as a potential biomarker of asthma severity. Several studies also demonstrated the role of BDNF and other neurotrophins in the other chronic respiratory diseases, for example, chronic obstructive pulmonary disease.17 Moreover, the positive therapeutic effect of different anti-inflammatory antiasthmatic medicaments is probably mediated through the neurotrophins regulation.18 A recent study has demonstrated that the monotherapy with salmeterol increased BDNF concentration in serum and platelets which may explain the adverse effect of such monotherapy on the asthmatic airways. The adding of fluticasone inhibited this increase.19 Besides the studies reporting the association between the expression and production of BDNF and various aspects of allergic presentation, there are only a few Pediatric Pulmonology
reports on the association between genetic variations in the BDNF gene and allergic diseases. Hence, we studied the functional polymorphism Val66Met (also referred to as rs6265 or G196A) in the BDNF gene in a group of asthmatic children and healthy controls. SUBJECTS AND METHODS Study Population
We studied 248 asthmatic patients (141 boys and 107 girls with an average age of 12.28 0.24 years; age range 5–17 years), who were recruited from the inpatient wards at the Department of Paediatrics, University Hospital Martin (Slovak Republic), over a period of 12 months. Bronchial asthma was defined as recurrent airway obstruction manifested by wheezing and dyspnoea that was relieved spontaneously or by bronchodilatator therapy (as defined in the Global Initiative for Asthma). The diagnosis of bronchial asthma was based on the history of wheeze or cough during the last 12 months, use of any asthma medication in the last 12 months, and a doctor’s diagnosis of asthma ever. The children suffering from any serious internal diseases (immune, cardiovascular, gastrointestinal, neurological, and respiratory) except of bronchial asthma were not enrolled into the study. The children suffering from acute respiratory infection or with the history of infection during the last 4 weeks were also excluded from the study. None of the subjects smoked and the children exposed to passive smoking were excluded from the study. Healthy subjects were represented by 249 children of comparable age and without any allergic disease or subclinical atopy (117 boys and 132 girls with an average age of 13.14 0.48 years), who were recruited during regular preventive examinations by general practitioners. The study was approved by the Ethics Committee of the Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava. DNA samples used in this study were collected from Slovak participants following standard protocols. Informed consent was obtained from the parents of all the participating children. Skin Prick Testing
In all children, we performed skin prick tests (SPT) with a panel of common inhalant allergens (Dermatophagoides pteronyssinus; Dermatophagoides farinae; cat dander; dog dander; mixed cereals; mixed grasses; mixed moulds; and mixed trees) (ALK-ABELLO, Hørsholm, Denmark). A skin prick test with allergens was defined as positive if the mean of the longest diameter and the diameter perpendicular to it and its mid-point were 3 mm. All the tests were performed by the same trained operator. Those children with at least one positive SPT were considered atopic.
BDNF Polymorphism in Children with Asthma
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DNA Extraction
Statistical Analysis
DNA was extracted from peripheral blood leukocytes, using a commercial procedure (Wizard Genomic DNA Purification Kit, Promega, Madison, WI).
The association of the BDNF gene polymorphism Val66Met with bronchial asthma was determined by Pearson’s chi-square (x2) test or Fisher’s exact test. Odds ratios (OR) and confidence intervals (95% CI) were used to analyse the frequencies of the genotypes in patients with bronchial asthma compared to the group of healthy children. P values less than or equal to 0.05 were considered statistically significant.
Polymerase Chain Reaction and Endonuclease Restriction Digestion
Polymerase chain reaction (PCR) was used to determine the BDNF Val66Met (rs6265, G196A) single nucleotide polymorphism (SNP) in a 304-bp fragment. The gene was amplified by PCR using the following primer pairs: sense 50 -AAACATCCGAGGACAAGGTG30 and antisense 50 -CCTCATGGACATGTTTGCAG-30 . PCR was performed as follows: initial 5 min activation at 958C followed by 35 cycles of denaturation (958C, 30 sec), annealing (608C, 30 sec), and extension (728C, 1 min), and a final extension at 728C for 7 min. After PCR, 5 ml of DNA products were separated by electrophoresis on a 2% agarose gel and visualized by a UV illuminator to detect fragments with a length of 304 bp. PCR products were digested with 1U of the fast digest restriction enzyme PmlI (Fermentas, Waltham, MA) at 378C for 5 min. DNA fragments were separated by electrophoresis on 2% agarose gels and visualized by a UV illuminator to measure DNA fragment lengths. Three possible genotypes were observed: Val/Val 124 bp, 180 bp; Val/Met 124 bp, 180 bp, 304 bp; and Met/Met 304 bp.
RESULTS
We examined 248 children with bronchial asthma (141 boys and 107 girls with an average age of 12.28 0.24 years) and 249 healthy subjects (117 boys and 132 girls with an average age of 13.14 0.48 years). Both groups were comparable for age and gender. The demographic characteristics of the studied subjects are reported in Table 1. The frequencies of genotypes and alleles in asthmatics and healthy children are shown in Table 2. The distribution of genotypes was tested for deviation from Hardy– Weinberg equilibrium. Tests for derivation from Hardy– Weinberg equilibrium in case were in equilibrium P ¼ 0.863 and in consequences of low present variant homozygote in healthy subjects P < 0.05. The differences are consequences of low present variant homozygote in healthy subjects. The BDNF Val66Met polymorphism is
TABLE 1— General Characteristics of the Study Population Parameter Total number Male Female Age (years) Age range (years) Atopy Family history for asthma Family history for atopy Age of diagnosis of BA Controlled asthma Uncontrolled asthma Treatment ICS LTRA ICS þ LABA Antihistamines Compliance with therapy Allergy markers Total IgE (IU/ml) Eosinophils (109/L) Lung function test FEV1 (%predicted) PEF25–75% (%predicted)
Asthmatic subjects (N/%)
Healthy subjects (N/%)
P-value
248 (100) 141 (56.9) 107 (43.1) 12.28 0.24 5–18 170 (68.5) 92 (37.1) 133 (53.6) 7.14 2.89 216 (87.0) 32 (13.0)
249 (100) 117 (47.0) 132 (53.0) 13.14 0.48 5–18 — 19 (7.6) 37 (14.9) — — —
— n.s. n.s. n.s. n.s. — P < 0.001 P < 0.001 — — —
84 (33.9) 93 (37.5) 113 (45.6) 226 (91.1) 228 (91.9)
— — — — —
— — — — —
235.13 24.87 0.40 0.10
74.77 22.71 0.14 0.02
P < 0.001 P ¼ 0.009
94.37 0.99 88.95 1.98
98.04 1.54 97.92 3.12
P < 0.05 P < 0.01
BA, bronchial asthma; FEV1, forced expiratory volume in 1 sec; ICS, inhaled corticosteroid; LABA, long-acting beta-2-agonist; LTRA, leukotriene receptor antagonist; n.s., not significant; PEF, peak expiratory flow; ppb, parts per billion; ppm, parts per million.
Pediatric Pulmonology
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TABLE 2— Distribution of Genotypes and Alleles of Brain-Derived Neurotrophic Factor (BDNF) and the Risk of Developing Bronchial Asthma BDNF A/G Val66Met
Healthy subjects N (%)
GG Val/Val GA Val/Met AA Met/Met GA þ AA Val/Met þ Met/Met Allele G (Val) Allele A (Met)
117 129 3 132 363 135
Asthmatic subjects N (%)
(47) (51.8) (1.2) (53) (72.9) (27.1)
153 83 12 95 389 107
(61.7) (33.5) (4.8) (38.3) (78.4) (21.6)
OR (95% CI)
P-value
Chi-square
Reference 0.69 (0.48–0.99) 4.17 (1.16–14.96) 0.81 (0.57–1.16) 1.35 (1.01–1.81) 0.74 (0.55–0.99)
— 0.045 0.018 0.25 0.05 0.05
— 4 5.61 1.32 4.13 4.13
OR, odds ratio; CI, confidence interval.
highly heterogeneous among the different ethnic populations, even within Europe and variant homozygote genotype is in very low frequency in Caucasian population. We observed only 3.5% of variant homozygote in our other study.20 The prevalence of the Val66Met polymorphisms (rs6265) in the BDNF gene among the 248 asthmatic children were: 61.7% for Val/Val homozygosity, 33.5% for Val/Met heterozygosity and 4.8% for Met/Met homozygosity (Table 2). The distributions of the genotypes in the healthy subjects were: 47.0% Val/Val, 51.8% Val/Met, and 1.2% Met/Met. The corresponding allele frequencies are shown in Table 2. The presence of the variant genotype (AA, Met/Met) may represent a risk factor for asthma (OR ¼ 4.17, 95% CI ¼ 1.16–14.96, P ¼ 0.018), whereas the heterozygous genotype (Val/Met) was a positive protective genotype (OR ¼ 0.69, 95% CI ¼ 0.48–0.99, P ¼ 0.045). When we tested only homozygous genotype then again variant homozygote may represent potential risk factor with borderline statistical significance (OR ¼ 3.059, 95% CI ¼ 0.844–11.088, P ¼ 0.07). Regarding the subjects with Met/Met genotype, there were 5 females (41.7%) among asthmatics and 3 females (100%) among healthy controls. The Met/Met subjects did not differed in age, but yielded significantly higher values of total IgE and peripheral blood eosinophils when comparing asthmatics to controls. The G-allele was
the most common allele in children with bronchial asthma (78.4%) and healthy children (72.9%). When analysis was performed according to the gender of the studied subjects, the variant genotype was a risk factor for bronchial asthma (Table 3) in both boys and girls, but because of the low frequencies of the variant genotype, it was impossible to calculate statistical parameters. The heterozygous genotype was a positive protective factor in only the girls (OD ¼ 0.34, 95% CI ¼ 0.20–0.59, P ¼ 0.001, chisquare ¼ 15.48). DISCUSSION
The association between genetic variation in the BDNF gene and bronchial asthma is controversial. We studied the SNP Val66Met in the BDNF gene in a group of asthmatic children and healthy controls. We found that children with bronchial asthma displayed a higher prevalence of the homozygous variants (AA, Met/Met) compared to healthy subjects. Regarding the gender of the studied subjects, we were unable to detect any significant differences in allele distribution, although the heterozygous form showed a slightly protective effect in girls. There are only a few reports on the possible interaction between genetic variation in the BDNF gene and bronchial asthma. Andiappan et al.21 studied the relationship between
TABLE 3— Distribution of the Genotypes of Brain-Derived Neurotrophic Factor (BDNF) and the Risk of Developing Bronchial Asthma in Boys and Girls BDNF A/G Val66Met
Healthy subjects boys N (%)
Asthmatic subjects boys N (%)
OR (95% CI)
P-value
Chi-square
GG Val/Val GA Val/Met AA Met/Met Allele G (Val) Allele A (Met)
57 (48.7) 60 (51.3) 0 (0) 174 (74.4) 60 (25.6) Healthy subjects girls N (%) 60 (45.5) 69 (52.3) 3 (2.3) 189 (71.6) 75 (28.4)
80 (56.7) 54 (38.3) 7 (5) 214 (75.9) 68 (24.1) Asthmatic subjects girls N (%) 73 (68.2) 29 (27.1) 5 (4.7) 175 (81.8) 39 (18.2)
Reference 0.59 (0.36–0.07) n.d. 1.08 (0.73–1.62) 0.92 (0.62–1.38) OR (95% CI) Reference 0.34 (0.20–0.59) 2.11 (0.49–9.03) 1.78 (1.15–2.76) 0.56 (0.36–0.87)
— 0.04 n.d. 0.69 0.69 P — 0.001 n.d. 0.01 0.01
— 4.37 n.d. 0.16 0.16 Chi-square — 15.48 n.d. 6.75 6.75
GG Val/Val GA Val/Met AA Met/Met Allele G (Val) Allele A (Met)
OR, odds ratio; CI, confidence interval; n.d., not determined.
Pediatric Pulmonology
BDNF Polymorphism in Children with Asthma
SNPs in the BDNF gene and genetic predisposition to allergic rhinitis and bronchial asthma in an ethnic Chinese population of Singapore. Their results identified a significant association for the tag SNP rs10767664 with allergic rhinitis (P ¼ 0.0007, OR 1.3) or bronchial asthma (P ¼ 0.0005, OR 1.3), using a dominant model of association. The SNP rs10767664 was strongly linked to the functional polymorphism rs6265, which was also investigated in our study. This polymorphism also affects BDNF expression.21 In another study, a multi-locus analysis of several candidate genes involved in neurogenic inflammation in pediatric asthma was performed. In that study, the BDNF SNP rs7124442 correlated with increased levels of exhaled nitric oxide, but was not associated with asthma susceptibility. However, other results from that study confirmed that neurogenic inflammation contributed to bronchial asthma.22 Another important study from Germany investigated whether polymorphisms in the BDNF gene influenced the development or severity of asthma and other atopic diseases. Nine different polymorphisms with minor allele frequency were identified and only one of them elicited an amino acid change from valine to methionine. In the cross-sectional study population, no significant association was found with asthma or any atopic disease. However, when more severe asthma patients were analyzed, a significant association with three polymorphisms (rs6265, rs11030101, and rs11030100) was found. The authors of this report suggested that certain BDNF polymorphisms may contribute to severe forms of asthma.23 In a recent study, the rs6265 and rs12206221 polymorphisms were significantly associated with asthma in children.24 However, in another study, BDNF gene SNPs (four selected polymorphisms: rs6265, rs2030324, rs988748, and rs7124442) did not significantly affect asthma susceptibility or severity.25 Furthermore, in a small group of asthmatic children, there was no association of the rs6265 polymorphism with asthma. However, the Val allele was much more frequent in male asthmatic patients. On the other hand, for the 270C/T polymorphism, there was a significant difference between asthmatic patients and healthy controls.26 Another study by the same authors further confirmed the lack of association between rs6265 and asthma.27 The data from some studies are in concordance with our results, whereas the others are not. Our study has several limitations such as relatively small sample size of the studied subjects and the absence of BDNF concentration measurement in the serum. Comparing the sample size with the other existing studies about the BDNF polymorphisms and asthma, the numbers are similar (with exception of the study with 655 asthmatics23). Our results are comparable to the results from the other geographical settings, where also the association between functional polymorphism (rs6265) in the BDNF gene was also described in conjunction with bronchial asthma.21,23,24 Three other studies conducted by the same authors on the
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same population on the other hand did not confirm this association,25–27 although Val allele was more frequent in male asthmatics.27 The similar prevalence of variant homozygote we observed also in another our study with the different patients (depressed patients), but the association was not confirmed what could suggest the specific link between BDNF gene, neurogenic inflammation, and bronchial asthma.20 Due to relatively small sample size it is not possible to draw a strong conclusions, however, our results supports in concordance with some other few available studies the possible link between the BDNF gene and asthma susceptibility. The significance of the Val66Met substitution in the prodomain of BDNF is still not clear. Egan et al.28 showed that the Val66Met (rs6265) polymorphism did not affect the function of the mature BDNF protein, but it did significantly alter the intracellular trafficking and packaging of pro-BDNF. The Val allele could probably be associated with increased activity in this respect and therefore, the homozygous Val/Val genotype can cause increased BDNF activity, thereby enhancing airflow limitation and airway hyperresponsiveness.29 According to our results, the association of the homozygous Met/Met genotype with bronchial asthma could lead to the decreased production of BDNF. It was previously proposed that BDNF exerts some beneficial activity in the lungs and pulmonary vasculature; therefore, its increased secretion and activity cannot be definitively described as a universal risk factor for the development of allergic diseases.30 In a small group of asthmatic children, there was a higher frequency of the Val allele in boys than in girls,26 which is consistent with the observation that boys are more susceptible to asthma.31 However, we did not detect any significant differences in allele distribution between the genders. The association between BDNF gene polymorphism and other atopic diseases has also been studied. Ma et al.32 found significant differences for the 270C/T polymorphism in the C/T genotype distribution and T allele frequencies between atopic dermatitis patients and healthy controls. Moreover, serum levels of BDNF correlated with atopic dermatitis severity. The authors concluded that the T allele in the 270C/T genotype could be a risk factor for atopic dermatitis, especially in the intrinsic form and male atopic dermatitis. However, in another study, no association between functional polymorphism (Val66Met) and atopic dermatitis was observed.33 The genetic variations in the BDNF gene could influence the expression of BDNF in the airways; however, we did not perform this examination, which was a weakness of our study. Nevertheless, the role of BDNF expression and concentration has been studied in several other animal and human studies. Transfected cells Pediatric Pulmonology
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with Met allele showed lower depolarization-induced secretion of BDNF, while constitutive secretion was unchanged and the heterozygote co-expression resulted in decreased secretion of BDNF.29 Since also the heterozygous genotype is able to influence the secretion of BDNF, therefore we chose the heterozygous model for statistical analyses. The increased levels of BDNF were described by several authors, but usually the significant changes were found only in moderate and severe asthmatics.16 The BDNF concentration has been reported in increased concentration in blood and also locally in asthmatic airways, especially after allergen exposure.10,34 Another important fact is that Met allele may be responsible for increased sensitivity to stress, for example, oxidative. We estimated the markers of oxidative damage (protein and lipid) in our studied population and we observed significantly elevated oxidative damage in association with asthma.35 When we analyzed the oxidative damage according to the BDNF genotype (data not shown), we found higher oxidative damage in patients with variant Met/Met genotype compared to other BDNF genotypes. Our study has shown that functional polymorphism (rs6265) in the BDNF gene is associated with bronchial asthma. This finding contributes to the evidence supporting the positive association between BDNF gene variations and asthma susceptibility in children. ACKNOWLEDGMENT
We thank J. Bencatova, Z. Cetlova, and A. Kempna for their laboratory assistance. The project was supported by grants from the Ministry of Education, Science, Research and Sport of the Slovak Republic VEGA 1/0071/11.
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8. Path G, Braun A, Meents N, Kerzel S, Quarcoo D, Raap U, Hoyle GW, Nockher WA, Renz H. Augmentation of allergic early-phase reaction by nerve growth factor. Am J Respir Crit Care Med 2002;1066:818–826. 9. Braun A, Lommatzsch M, Neuhaus-Steinmetz U, Quarcoo D, Glaab T, McGregor GP, Fischer A, Renz H. Brain-derived neurotrophic factor (BDNF) contributes to neuronal dysfunction in a model of allergic airway inflammation. Br J Pharmacol 2004;141:431–440. 10. Virchow JC, Julius P, Lommatzsch M, Luttmann W, Renz H, Braun A. Neurotrophins are increased in bronchoalveolar lavage fluid after segmental allergen provocation. Am J Respir Crit Care Med 1998;158:2002–2005. 11. Wetmore C, Olson L. Neuronal and nonneuronal expression of neurotrophins and their receptors in sensory and sympathetic ganglia suggest new intercellular trophic interactions. J Comp Neurol 1995;353:143–159. 12. Joos GF, Germonpre PR, Pauwels RA. Role of tachykinins in asthma. Allergy 2000;55:321–337. 13. Lewin GR, Barde YA. Physiology of neurotrophins. Annu Rev Neurosci 1996;19:289–317. 14. Aravamudan B, Thompson M, Pabelick C, Prakash YS. Brainderived neurotrophic factor induces proliferation of human airway smooth muscle cells. J Cell Mol Med 2012;16:812–823. 15. Sathish V, Vanoosten SK, Miller BS, Aravamudan B, Thompson MA, Pabelick CM, Vassallo R, Prakash YS. Brain-derived neurotrophic factor in cigarette smoke-induced airway hyperreactivity. Am J Respir Cell Mol Biol 2013;48:431–438. 16. Muller GC, Pitrez PM, Teixeira AL, Pires PS, Jones MH, Stein RT, Bauer ME. Plasma brain-derived neutrophic factor levels are associated with clinical severity in school age children with asthma. Clin Exp Allergy 2010;40:1755–1759. 17. Stoll P, Wuertemberger U, Bratke K, Zingler C, Virchow JC, Lommatzsch M. Stage-dependent association of BDNF and TGF-b1 with lung function in stable COPD. Respir Res 2012;13:116. 18. Chang R-S, Wang S-D, Wang Y-C, Lin L-J, Kao S-T, Wanf J-Y. Xiao-Qing-Long-Tang shows preventive effect of asthma in an allergic asthma mouse model trough neurotrophin regulation. BMC Complement Altern Med 2013;13:220. 19. Lommatzsch N, Lindner Y, Edner A, Bratke K, Kuepper M, Virchow JC. Adverse effects of salmeterol in asthma: A neuronal perspective. Thorax 2009;64:763–769. 20. Evinova A, Babusikova E, Straka S, Ondrejka I, Lehotsky J. Analysis of genetic polymorphisms of brain-derived neurotrophic factor and methylenetetrahydrofolate reductase in depressed patients in a Slovak (Caucasian) population. Gen Physiol Biophys 2012;31:415–422. 21. Andiappan AK, Parate PN, Anatharaman R, Suri BK, Wang de Y, Chew FT. Genetic variation on BDNF is associated with allergic asthma and allergic rhinitis in an ethnic Chinese population in Singapore. Cytokine 2011;56:218–223. 22. Szczepankiewicz A, Sobkowiak P, Rachel M, Breborowicz A, Schoneich N, Bruce K, Kycler Z, Wojsyk-Banaszak I, DmitrzakWeglarz M. Multilocus analysis of candidate genes involved in neurogenic inflammation in pediatric asthma and related phenotypes: A case-control study. J Asthma 2012;49:329– 335. 23. Zeilinger S, Pinto LA, Nockher WA, Depner M, Klopp N, Illig T, von Mutius E, Renz H, Kabesch M. The effect of BDNF gene variants on asthma in German children. Allergy 2009;64:1790– 1794. 24. Yinli C, Jie H, Jun G, Peiling L, Weihong Y. Association between brain-derived neurotrophic factor variants and asthma in Chinese Han children. Acta Paediatr 2013;102:e247–e250.
BDNF Polymorphism in Children with Asthma 25. Szczepankiewicz A, Rose-Zerilli MJ, Barton SJ, Holgate ST, Holloway JW. Association analysis of brain-derived neurotrophic factor gene polymorphisms in asthmatic families. Int Arch Allergy Immunol 2009;149:343–349. 26. Szczepankiewicz A, Breborowicz A, Skibinska M, Wilkosc M, Tomaszewska M, Hauser J. Association analysis of brain-derived neurotrophic factor gene polymorphisms in asthmatic children. Pediatr Allergy Immunol 2007;18:293–297. 27. Szczepankiewicz A, Rachel M, Sobkowiak P, Kycler Z, WojsykBanaszak I, Schoneich N, Szczawinska-Poplonyk A, Breborowicz A. Neutrotrophin serum concentrations and polymorphisms of neurotrophins and their receptors in children with asthma. Respir Med 2013;107:30–36. 28. Egan MF, Weinberger DR, Lu B. Schizophrenia, III: brain-derived neurotrophic factor and genetic risk. Am J Psychiatry 2003;160: 1242. 29. Egan MF, Kojima M, Callicott JH, Goldberg TE, Kolachana BS, Bertolino A, Zaitsev E, Goldman D, Dean M, Lu B, et al. The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function. Cell 2003;112:257–2269.
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30. Groneberg DA, Fischer TC, Peckenschneider N, Noha QT, Welte T, Welker P. Cell type-specific regulation of brain-derived neurotrophic factor in states of allergic inflammation. Clin Exp Allergy 2007;37:1386–1391. 31. Arshad SH, Kurukulaaratchy RJ, Fenn M, Matthews S. Early life risk factors for recurrent wheeze, asthma, and bronchial hyperresponsiveness at 10 years of age. Chest 2005;127:502–508. 32. Ma L, Gao XH, Zhao LP, Di ZH, Mchepange UO, Zhang L, Chen HD, Wei HC. Brain-derived neurotrophic factor gene polymorphisms and serum levels in Chinese atopic dermatitis patients. J Eur Acad Dermatol Venereol 2009;23:1277–1281. 33. Hoffjan S, Parwez Q, Petrasch-Parwez E, Stemmler S. Variation in the BDNF and NGFB genes in German atopic dermatitis patients. Mol Cell Probes 2009;23:35–38. 34. Noga O, Hanf G, Schaper C, O’Connor A, Kunkel G. The influence of inhalative corticosteroids on circulating nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in allergic asthmatics. Clin Exp Allergy 2001;31:1906–1912. 35. Babusikova E, Jesenak M, Kirschnerova R, Banovcin P, Dobrota D. Association of oxidative stress and GST-T1 gene with childhood bronchial asthma. J Physiol Pharmacol 2009;60:27–30.
Pediatric Pulmonology
Val66Met Polymorphism in the BDNF Gene in Children with Bronchial Asthma Milos Jesenak, MD, PhD,1 Eva Babusikova, MSc, PhD,2 Andrea Evinova, MSc, PhD,2 Peter Banovcin, MD, PhD,1 and Dusan Dobrota, MD, PhD2 Summary. Objectives: Bronchial asthma is a chronic respiratory disease characterized by airway inflammation. There is increasing evidence that neurotrophins play an important role in the development and maintenance of neurogenic airway inflammation in chronic allergic diseases. Working Hypothesis: Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and has several important functions in the airways. There are only a few reports on the association between genetic variations in the BDNF gene and various allergic diseases, and the results are generally conflicting. Therefore, we aimed to study the functional polymorphism Val66Met (also called rs6265 or G196A) in the BDNF gene in a group of asthmatic children and healthy controls. Study Design, Patient-Selection, and Methodology: We studied 248 asthmatic patients (aged 12.28 0.24 years) and 249 healthy children (aged 13.14 0.48 years). Analysis of the Val66Met polymorphism of the BDNF gene was performed by polymerase chain reaction (PCR) and PCR products were digested by PmlI. Results: The prevalence of the Val66Met polymorphisms (Val/Val, Val/Met, and Met/Met) was 61.7%, 33.5%, and 4.8% in asthmatics, respectively, and 47.0%, 51.8%, and 1.2% in healthy subjects, respectively. We observed a significant association of the Met/Met variant genotype with asthmatics (OR ¼ 4.17, 95% CI ¼ 1.16–14.96, P ¼ 0.018). The Val/Met genotype was protective against bronchial asthma (OR ¼ 0.69, 95% CI ¼ 0.48–0.99, P ¼ 0.045), especially in girls (OR ¼ 0.34, 95% CI ¼ 0.20–0.59, P ¼ 0.001). Conclusion: Specific BDNF gene polymorphism may contribute to bronchial asthma susceptibility. Our study suggested the positive association between selected functional BDNF ß 2014 Wiley Periodicals, Inc. polymorphism (rs6265) and asthma in children. Pediatr Pulmonol. Key words: brain-derived neurotrophic factor; bronchial asthma; children; neurogenic inflammation; neurotrophins; gene polymorphism. Funding source: Ministry of Education, Science, Research and Sport of the Slovak Republic VEGA; Number: 1/0071/11.
INTRODUCTION
Bronchial asthma is the most frequent chronic respiratory disease in children and is clinically characterized by airway inflammation, airway hyperresponsiveness, and reversible airflow limitations. However, the links between airway inflammation and changes in pulmonary functions are not completely understood. There is increasing evidence that one of the most important pathogenetic aspects responsible for the induction, maintenance, and progress of the airway changes in asthmatic patients is neurogenic inflammation.1 While there is convincing evidence of neurogenic inflammation in animal models of asthma,2 the evidence in humans is less clear and different studies brought conflicting and inconsistence results.3 Research was focused on the concentration of different neurotrophins and their function in the airways, but more recently, the association studies between different polymorphisms in the neurotrophins’ genes were also conducted. Neurotrophins play a critical role in the pathogenesis of allergic inflammation in asthma.4,5 ß 2014 Wiley Periodicals, Inc.
There is increasing evidence of the various members of the neurotrophin family participating in the development and
1 Department of Paediatrics, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia. 2 Department of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia.
Conflict of interest: None.
Correspondence to: Assoc. Prof. Eva Babusikova, MSc, PhD, Department of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Mala Hora 4, 036 01 Martin, Slovakia. E-mail: [email protected] Received 6 May 2013; Accepted 21 April 2014. DOI 10.1002/ppul.23065 Published online in Wiley Online Library (wileyonlinelibrary.com).
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maintenance of airway inflammation. There are several studies confirming the involvement of nerve growth factor in bronchial asthma,6–8 but the studies with brain-derived neurotrophic factor (BDNF) showed conflicting results regarding both the association between BDNF concentration or selected polymorphisms in asthmatic patients. In an animal model of allergic asthma, the up-regulation of BDNF in bronchoalveolar lavage fluid (BALF) and airway infiltrates after allergen challenge in sensitized mouse was detected.9 Up-regulation of neurotrophins including BDNF has also been demonstrated in asthmatics following segmental allergen provocation.10 Most of the vagal neurons innervating the lung are BDNF-dependent.11 Therefore, the local production of BDNF in allergic inflammation may be involved in neuronal changes occurring in bronchial asthma. Several authors have suggested that BDNF may serve as a mediator, linking airway inflammation with the neuronal changes observed in bronchial asthma. BDNF is a key mediator of neuronal plasticity, which significantly contributes to airway obstruction and hyperresponsiveness. Inhibition of endogenous BDNF reduces enhanced airway tone and neuronal hyperreactivity in allergen-challenged animals, while the administration of recombinant BDNF induces these asthmatic changes in healthy animals.9 Another possible mechanism of BDNF action in asthma is its stimulatory effect on the production and release of different tachykinins (e.g., substance P and neurokinin A), which are other very important mediators of asthma.12,13 BDNF enhances the proliferation of airway smooth muscles, which can contribute to the remodeling of the airways in chronic respiratory diseases.14 The expression of BDNF was shown to be induced also by cigarette smoke followed by the changes in the signaling in airway smooth muscle, which then contribute to cigarette smoke-induced airway hyperresponsiveness.15 Moreover, BDNF is known to elicit neuropeptide synthesis in neurons and support the survival, differentiation and function of a broad spectrum of neurons of the central and peripheral nervous system. At the clinical level, subjects with moderate to severe asthma have higher BDNF levels than mild asthmatics and controls16; therefore, BDNF could serve as a potential biomarker of asthma severity. Several studies also demonstrated the role of BDNF and other neurotrophins in the other chronic respiratory diseases, for example, chronic obstructive pulmonary disease.17 Moreover, the positive therapeutic effect of different anti-inflammatory antiasthmatic medicaments is probably mediated through the neurotrophins regulation.18 A recent study has demonstrated that the monotherapy with salmeterol increased BDNF concentration in serum and platelets which may explain the adverse effect of such monotherapy on the asthmatic airways. The adding of fluticasone inhibited this increase.19 Besides the studies reporting the association between the expression and production of BDNF and various aspects of allergic presentation, there are only a few Pediatric Pulmonology
reports on the association between genetic variations in the BDNF gene and allergic diseases. Hence, we studied the functional polymorphism Val66Met (also referred to as rs6265 or G196A) in the BDNF gene in a group of asthmatic children and healthy controls. SUBJECTS AND METHODS Study Population
We studied 248 asthmatic patients (141 boys and 107 girls with an average age of 12.28 0.24 years; age range 5–17 years), who were recruited from the inpatient wards at the Department of Paediatrics, University Hospital Martin (Slovak Republic), over a period of 12 months. Bronchial asthma was defined as recurrent airway obstruction manifested by wheezing and dyspnoea that was relieved spontaneously or by bronchodilatator therapy (as defined in the Global Initiative for Asthma). The diagnosis of bronchial asthma was based on the history of wheeze or cough during the last 12 months, use of any asthma medication in the last 12 months, and a doctor’s diagnosis of asthma ever. The children suffering from any serious internal diseases (immune, cardiovascular, gastrointestinal, neurological, and respiratory) except of bronchial asthma were not enrolled into the study. The children suffering from acute respiratory infection or with the history of infection during the last 4 weeks were also excluded from the study. None of the subjects smoked and the children exposed to passive smoking were excluded from the study. Healthy subjects were represented by 249 children of comparable age and without any allergic disease or subclinical atopy (117 boys and 132 girls with an average age of 13.14 0.48 years), who were recruited during regular preventive examinations by general practitioners. The study was approved by the Ethics Committee of the Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava. DNA samples used in this study were collected from Slovak participants following standard protocols. Informed consent was obtained from the parents of all the participating children. Skin Prick Testing
In all children, we performed skin prick tests (SPT) with a panel of common inhalant allergens (Dermatophagoides pteronyssinus; Dermatophagoides farinae; cat dander; dog dander; mixed cereals; mixed grasses; mixed moulds; and mixed trees) (ALK-ABELLO, Hørsholm, Denmark). A skin prick test with allergens was defined as positive if the mean of the longest diameter and the diameter perpendicular to it and its mid-point were 3 mm. All the tests were performed by the same trained operator. Those children with at least one positive SPT were considered atopic.
BDNF Polymorphism in Children with Asthma
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DNA Extraction
Statistical Analysis
DNA was extracted from peripheral blood leukocytes, using a commercial procedure (Wizard Genomic DNA Purification Kit, Promega, Madison, WI).
The association of the BDNF gene polymorphism Val66Met with bronchial asthma was determined by Pearson’s chi-square (x2) test or Fisher’s exact test. Odds ratios (OR) and confidence intervals (95% CI) were used to analyse the frequencies of the genotypes in patients with bronchial asthma compared to the group of healthy children. P values less than or equal to 0.05 were considered statistically significant.
Polymerase Chain Reaction and Endonuclease Restriction Digestion
Polymerase chain reaction (PCR) was used to determine the BDNF Val66Met (rs6265, G196A) single nucleotide polymorphism (SNP) in a 304-bp fragment. The gene was amplified by PCR using the following primer pairs: sense 50 -AAACATCCGAGGACAAGGTG30 and antisense 50 -CCTCATGGACATGTTTGCAG-30 . PCR was performed as follows: initial 5 min activation at 958C followed by 35 cycles of denaturation (958C, 30 sec), annealing (608C, 30 sec), and extension (728C, 1 min), and a final extension at 728C for 7 min. After PCR, 5 ml of DNA products were separated by electrophoresis on a 2% agarose gel and visualized by a UV illuminator to detect fragments with a length of 304 bp. PCR products were digested with 1U of the fast digest restriction enzyme PmlI (Fermentas, Waltham, MA) at 378C for 5 min. DNA fragments were separated by electrophoresis on 2% agarose gels and visualized by a UV illuminator to measure DNA fragment lengths. Three possible genotypes were observed: Val/Val 124 bp, 180 bp; Val/Met 124 bp, 180 bp, 304 bp; and Met/Met 304 bp.
RESULTS
We examined 248 children with bronchial asthma (141 boys and 107 girls with an average age of 12.28 0.24 years) and 249 healthy subjects (117 boys and 132 girls with an average age of 13.14 0.48 years). Both groups were comparable for age and gender. The demographic characteristics of the studied subjects are reported in Table 1. The frequencies of genotypes and alleles in asthmatics and healthy children are shown in Table 2. The distribution of genotypes was tested for deviation from Hardy– Weinberg equilibrium. Tests for derivation from Hardy– Weinberg equilibrium in case were in equilibrium P ¼ 0.863 and in consequences of low present variant homozygote in healthy subjects P < 0.05. The differences are consequences of low present variant homozygote in healthy subjects. The BDNF Val66Met polymorphism is
TABLE 1— General Characteristics of the Study Population Parameter Total number Male Female Age (years) Age range (years) Atopy Family history for asthma Family history for atopy Age of diagnosis of BA Controlled asthma Uncontrolled asthma Treatment ICS LTRA ICS þ LABA Antihistamines Compliance with therapy Allergy markers Total IgE (IU/ml) Eosinophils (109/L) Lung function test FEV1 (%predicted) PEF25–75% (%predicted)
Asthmatic subjects (N/%)
Healthy subjects (N/%)
P-value
248 (100) 141 (56.9) 107 (43.1) 12.28 0.24 5–18 170 (68.5) 92 (37.1) 133 (53.6) 7.14 2.89 216 (87.0) 32 (13.0)
249 (100) 117 (47.0) 132 (53.0) 13.14 0.48 5–18 — 19 (7.6) 37 (14.9) — — —
— n.s. n.s. n.s. n.s. — P < 0.001 P < 0.001 — — —
84 (33.9) 93 (37.5) 113 (45.6) 226 (91.1) 228 (91.9)
— — — — —
— — — — —
235.13 24.87 0.40 0.10
74.77 22.71 0.14 0.02
P < 0.001 P ¼ 0.009
94.37 0.99 88.95 1.98
98.04 1.54 97.92 3.12
P < 0.05 P < 0.01
BA, bronchial asthma; FEV1, forced expiratory volume in 1 sec; ICS, inhaled corticosteroid; LABA, long-acting beta-2-agonist; LTRA, leukotriene receptor antagonist; n.s., not significant; PEF, peak expiratory flow; ppb, parts per billion; ppm, parts per million.
Pediatric Pulmonology
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TABLE 2— Distribution of Genotypes and Alleles of Brain-Derived Neurotrophic Factor (BDNF) and the Risk of Developing Bronchial Asthma BDNF A/G Val66Met
Healthy subjects N (%)
GG Val/Val GA Val/Met AA Met/Met GA þ AA Val/Met þ Met/Met Allele G (Val) Allele A (Met)
117 129 3 132 363 135
Asthmatic subjects N (%)
(47) (51.8) (1.2) (53) (72.9) (27.1)
153 83 12 95 389 107
(61.7) (33.5) (4.8) (38.3) (78.4) (21.6)
OR (95% CI)
P-value
Chi-square
Reference 0.69 (0.48–0.99) 4.17 (1.16–14.96) 0.81 (0.57–1.16) 1.35 (1.01–1.81) 0.74 (0.55–0.99)
— 0.045 0.018 0.25 0.05 0.05
— 4 5.61 1.32 4.13 4.13
OR, odds ratio; CI, confidence interval.
highly heterogeneous among the different ethnic populations, even within Europe and variant homozygote genotype is in very low frequency in Caucasian population. We observed only 3.5% of variant homozygote in our other study.20 The prevalence of the Val66Met polymorphisms (rs6265) in the BDNF gene among the 248 asthmatic children were: 61.7% for Val/Val homozygosity, 33.5% for Val/Met heterozygosity and 4.8% for Met/Met homozygosity (Table 2). The distributions of the genotypes in the healthy subjects were: 47.0% Val/Val, 51.8% Val/Met, and 1.2% Met/Met. The corresponding allele frequencies are shown in Table 2. The presence of the variant genotype (AA, Met/Met) may represent a risk factor for asthma (OR ¼ 4.17, 95% CI ¼ 1.16–14.96, P ¼ 0.018), whereas the heterozygous genotype (Val/Met) was a positive protective genotype (OR ¼ 0.69, 95% CI ¼ 0.48–0.99, P ¼ 0.045). When we tested only homozygous genotype then again variant homozygote may represent potential risk factor with borderline statistical significance (OR ¼ 3.059, 95% CI ¼ 0.844–11.088, P ¼ 0.07). Regarding the subjects with Met/Met genotype, there were 5 females (41.7%) among asthmatics and 3 females (100%) among healthy controls. The Met/Met subjects did not differed in age, but yielded significantly higher values of total IgE and peripheral blood eosinophils when comparing asthmatics to controls. The G-allele was
the most common allele in children with bronchial asthma (78.4%) and healthy children (72.9%). When analysis was performed according to the gender of the studied subjects, the variant genotype was a risk factor for bronchial asthma (Table 3) in both boys and girls, but because of the low frequencies of the variant genotype, it was impossible to calculate statistical parameters. The heterozygous genotype was a positive protective factor in only the girls (OD ¼ 0.34, 95% CI ¼ 0.20–0.59, P ¼ 0.001, chisquare ¼ 15.48). DISCUSSION
The association between genetic variation in the BDNF gene and bronchial asthma is controversial. We studied the SNP Val66Met in the BDNF gene in a group of asthmatic children and healthy controls. We found that children with bronchial asthma displayed a higher prevalence of the homozygous variants (AA, Met/Met) compared to healthy subjects. Regarding the gender of the studied subjects, we were unable to detect any significant differences in allele distribution, although the heterozygous form showed a slightly protective effect in girls. There are only a few reports on the possible interaction between genetic variation in the BDNF gene and bronchial asthma. Andiappan et al.21 studied the relationship between
TABLE 3— Distribution of the Genotypes of Brain-Derived Neurotrophic Factor (BDNF) and the Risk of Developing Bronchial Asthma in Boys and Girls BDNF A/G Val66Met
Healthy subjects boys N (%)
Asthmatic subjects boys N (%)
OR (95% CI)
P-value
Chi-square
GG Val/Val GA Val/Met AA Met/Met Allele G (Val) Allele A (Met)
57 (48.7) 60 (51.3) 0 (0) 174 (74.4) 60 (25.6) Healthy subjects girls N (%) 60 (45.5) 69 (52.3) 3 (2.3) 189 (71.6) 75 (28.4)
80 (56.7) 54 (38.3) 7 (5) 214 (75.9) 68 (24.1) Asthmatic subjects girls N (%) 73 (68.2) 29 (27.1) 5 (4.7) 175 (81.8) 39 (18.2)
Reference 0.59 (0.36–0.07) n.d. 1.08 (0.73–1.62) 0.92 (0.62–1.38) OR (95% CI) Reference 0.34 (0.20–0.59) 2.11 (0.49–9.03) 1.78 (1.15–2.76) 0.56 (0.36–0.87)
— 0.04 n.d. 0.69 0.69 P — 0.001 n.d. 0.01 0.01
— 4.37 n.d. 0.16 0.16 Chi-square — 15.48 n.d. 6.75 6.75
GG Val/Val GA Val/Met AA Met/Met Allele G (Val) Allele A (Met)
OR, odds ratio; CI, confidence interval; n.d., not determined.
Pediatric Pulmonology
BDNF Polymorphism in Children with Asthma
SNPs in the BDNF gene and genetic predisposition to allergic rhinitis and bronchial asthma in an ethnic Chinese population of Singapore. Their results identified a significant association for the tag SNP rs10767664 with allergic rhinitis (P ¼ 0.0007, OR 1.3) or bronchial asthma (P ¼ 0.0005, OR 1.3), using a dominant model of association. The SNP rs10767664 was strongly linked to the functional polymorphism rs6265, which was also investigated in our study. This polymorphism also affects BDNF expression.21 In another study, a multi-locus analysis of several candidate genes involved in neurogenic inflammation in pediatric asthma was performed. In that study, the BDNF SNP rs7124442 correlated with increased levels of exhaled nitric oxide, but was not associated with asthma susceptibility. However, other results from that study confirmed that neurogenic inflammation contributed to bronchial asthma.22 Another important study from Germany investigated whether polymorphisms in the BDNF gene influenced the development or severity of asthma and other atopic diseases. Nine different polymorphisms with minor allele frequency were identified and only one of them elicited an amino acid change from valine to methionine. In the cross-sectional study population, no significant association was found with asthma or any atopic disease. However, when more severe asthma patients were analyzed, a significant association with three polymorphisms (rs6265, rs11030101, and rs11030100) was found. The authors of this report suggested that certain BDNF polymorphisms may contribute to severe forms of asthma.23 In a recent study, the rs6265 and rs12206221 polymorphisms were significantly associated with asthma in children.24 However, in another study, BDNF gene SNPs (four selected polymorphisms: rs6265, rs2030324, rs988748, and rs7124442) did not significantly affect asthma susceptibility or severity.25 Furthermore, in a small group of asthmatic children, there was no association of the rs6265 polymorphism with asthma. However, the Val allele was much more frequent in male asthmatic patients. On the other hand, for the 270C/T polymorphism, there was a significant difference between asthmatic patients and healthy controls.26 Another study by the same authors further confirmed the lack of association between rs6265 and asthma.27 The data from some studies are in concordance with our results, whereas the others are not. Our study has several limitations such as relatively small sample size of the studied subjects and the absence of BDNF concentration measurement in the serum. Comparing the sample size with the other existing studies about the BDNF polymorphisms and asthma, the numbers are similar (with exception of the study with 655 asthmatics23). Our results are comparable to the results from the other geographical settings, where also the association between functional polymorphism (rs6265) in the BDNF gene was also described in conjunction with bronchial asthma.21,23,24 Three other studies conducted by the same authors on the
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same population on the other hand did not confirm this association,25–27 although Val allele was more frequent in male asthmatics.27 The similar prevalence of variant homozygote we observed also in another our study with the different patients (depressed patients), but the association was not confirmed what could suggest the specific link between BDNF gene, neurogenic inflammation, and bronchial asthma.20 Due to relatively small sample size it is not possible to draw a strong conclusions, however, our results supports in concordance with some other few available studies the possible link between the BDNF gene and asthma susceptibility. The significance of the Val66Met substitution in the prodomain of BDNF is still not clear. Egan et al.28 showed that the Val66Met (rs6265) polymorphism did not affect the function of the mature BDNF protein, but it did significantly alter the intracellular trafficking and packaging of pro-BDNF. The Val allele could probably be associated with increased activity in this respect and therefore, the homozygous Val/Val genotype can cause increased BDNF activity, thereby enhancing airflow limitation and airway hyperresponsiveness.29 According to our results, the association of the homozygous Met/Met genotype with bronchial asthma could lead to the decreased production of BDNF. It was previously proposed that BDNF exerts some beneficial activity in the lungs and pulmonary vasculature; therefore, its increased secretion and activity cannot be definitively described as a universal risk factor for the development of allergic diseases.30 In a small group of asthmatic children, there was a higher frequency of the Val allele in boys than in girls,26 which is consistent with the observation that boys are more susceptible to asthma.31 However, we did not detect any significant differences in allele distribution between the genders. The association between BDNF gene polymorphism and other atopic diseases has also been studied. Ma et al.32 found significant differences for the 270C/T polymorphism in the C/T genotype distribution and T allele frequencies between atopic dermatitis patients and healthy controls. Moreover, serum levels of BDNF correlated with atopic dermatitis severity. The authors concluded that the T allele in the 270C/T genotype could be a risk factor for atopic dermatitis, especially in the intrinsic form and male atopic dermatitis. However, in another study, no association between functional polymorphism (Val66Met) and atopic dermatitis was observed.33 The genetic variations in the BDNF gene could influence the expression of BDNF in the airways; however, we did not perform this examination, which was a weakness of our study. Nevertheless, the role of BDNF expression and concentration has been studied in several other animal and human studies. Transfected cells Pediatric Pulmonology
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with Met allele showed lower depolarization-induced secretion of BDNF, while constitutive secretion was unchanged and the heterozygote co-expression resulted in decreased secretion of BDNF.29 Since also the heterozygous genotype is able to influence the secretion of BDNF, therefore we chose the heterozygous model for statistical analyses. The increased levels of BDNF were described by several authors, but usually the significant changes were found only in moderate and severe asthmatics.16 The BDNF concentration has been reported in increased concentration in blood and also locally in asthmatic airways, especially after allergen exposure.10,34 Another important fact is that Met allele may be responsible for increased sensitivity to stress, for example, oxidative. We estimated the markers of oxidative damage (protein and lipid) in our studied population and we observed significantly elevated oxidative damage in association with asthma.35 When we analyzed the oxidative damage according to the BDNF genotype (data not shown), we found higher oxidative damage in patients with variant Met/Met genotype compared to other BDNF genotypes. Our study has shown that functional polymorphism (rs6265) in the BDNF gene is associated with bronchial asthma. This finding contributes to the evidence supporting the positive association between BDNF gene variations and asthma susceptibility in children. ACKNOWLEDGMENT
We thank J. Bencatova, Z. Cetlova, and A. Kempna for their laboratory assistance. The project was supported by grants from the Ministry of Education, Science, Research and Sport of the Slovak Republic VEGA 1/0071/11.
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